The need for alternatives to petroleum resources for production of fuels and chemicals has become a major quest, generated from economic incentives associated with limited and diminishing supply (Kheshgi, H. S., R. C. Prince, and G. Marland. 2000. The potential of biomass fuels in the context of global climate change: focus on transportation fuels. Annu. Rev. Energy Environ. 25:199-244). The connection between increasing carbon dioxide and global warming has directed this quest toward formation of fermentation products derived from resources renewable through photosynthesis (McMillan J. D. (1997) Bioethanol production: status and prospects. Renew Energy 10:295-302). The development of yeast and bacterial biocatalysts has been applied to the commercial production of ethanol as an alternative fuel from starch and sucrose derived from commodity crops, e.g. corn and sugarcane (Dien, B. S., M. A. Cotta, and T. W. Jeffries. 2003. Bacteria engineered for fuel ethanol production: current status. Appl Microbiol Biotechnol 63:258-266). To expand production of ethanol and chemical feedstocks from renewable resources that do not economically impact these commodities, lignocellulosic resources, including forest and agricultural residues, have become targets for bioconversion cellulose and hemicellulose to fermentable sugars (Aden, A., M. Ruth, K. Ibsen, J. Jechura, K. Neeves, J. Sheehan, B. Wallace, L. Montague, A. Slayton, and J. Lukas. 2002. Lignocellulosic biomass to ethanol process design and economics utilizing co-current dilute acid prehydrolysis and enzymatic hydrolysis for corn stover. NREL/TP-510-32438. National Renewable Energy Laboratory, Golden, Colo. Available online at nrel.gov/docs/fy02osti/32438.pdf.
Cellulose comprises the major part of all plant biomass. The source of all cellulose is the structural tissue of plants. It occurs in close association with hemicellulose and lignin, which together comprise the major components of plant fiber cells. Cellulose consists of long chains of beta glucosidic residues linked through the 1,4 positions. These linkages cause the cellulose to have a high crystallinity and thus a low accessibility to enzymes or acid catalysts. Hemicellulose is an amorphous hetero-polymer which is easily hydrolyzed. Lignin, an aromatic three-dimensional polymer, is interspersed among the cellulose and hemicellulose within the plant fiber cell.
Previously reported processes for hydrolysing cellulose include biological and non-biological means of depolymerization. The biological methods involve the use a cellulase enzyme. The oldest and best known non-biological method of producing sugars from cellulose is the use of acid hydrolysis. The acid most commonly used in this process is sulfuric acid. In general, sulfuric acid hydrolysis can be categorized as either dilute acid hydrolysis or concentrated acid hydrolysis.
The dilute acid processes generally involve the use of 0.5% to 15% sulfuric acid to hydrolyze the cellulosic material. In addition, temperatures ranging from 90°-600° C., and pressure up to 800 psi are necessary to effect the hydrolysis. At high temperatures, the sugars degrade to form furfural and other undesirable by-products. The resulting glucose yields are generally low, less than 50%. Accordingly, the dilute acid processes have not been successful in obtaining sugars from cellulosic material in high yields at low cost.
In addition to these difficulties, it has been recognized that the fermentation of the sugars produced by dilute acid hydrolysis presents additional problems. The hydrolysis of cellulose and hemicellulose results in the production of pentose sugars for fermentation (Y. Y. Lee A1, Prashant Iyer, R. W. Torget. 1999. Dilute-Acid Hydrolysis of Lignocellulosic Biomass. Advances in Biochemical Engineering/Biotechnology Volume 65 pp. 93-115). The predominant structural polymer in the hemicellulose fraction of hardwoods and crop residues is methylglucuronoxylan (MeGAXn), a β-1,4 linked xylan in which xylose residues are periodically substituted with a-1,2-linked 4-O-methyl-glucuronic acid (Preston, J. F., J. C. Hurlbert, J. D. Rice, A. Ragunathan, and F. J. St. John. 2003. Microbial strategies for the depolymerization of glucuronoxylan: leads to biotechnological applications of endoxylanases, p. 191-210, Applications of Enzymes to Lignocellulosics. American Chemical Society, Washington D.C.). Resistance of the a-1,2 glucuronosyl linkages to dilute acid hydrolysis results in the release of methylglucuronoxylose (MeGAX), which is not fermented by bacterial biocatalysts currently used to convert hemicellulose to ethanol, e.g. E. coli KO11. The frequency of MeGAX substitutions on the xylose residues of methylglucuronoxylan ranges from less than one in ten in crop residues to one in six to seven in hardwoods, e.g. sweetgum, and as much as 21% of the carbohydrate may reside in this unfermentable fraction following dilute acid pretreatment (Maria E. Rodriguez, Alfredo Martinez, Lonnie Ingram, Keelnatham T Shamugam and James F Preston. 2001. Properties of the hemicellulose fractions of lignocellulosic biomass affecting bacterial ethanol production. ASM National Meeting, 2001.). As a result of the sometimes large yield of MeGAX following dilute acid processes, the sugar yield is low and fermentation is hampered in producing useful biofuels and chemical feedstocks from renewable photosynthetic resources.
Thus, there is an urgent need for an economically viable, environmentally safe microorganism that can ferment MeGAX resulting from dilute acid hydrolysis of photosynthetic resources to produce useful biofuels (such as ethanol) and chemical feedstocks (such as acetate).